This application is a 371 of PCT/SE99/01663, filed Sep. 23, 1999.
The present invention relates to methods for producing porous polysaccharide beads.
Polysaccharide gels are known to play an important role in the manufacture of materials for separation of mixtures of biomolecules. Among the characteristics making these gels interesting can be mentioned their inertness in contact with proteins and other biomolecules, and their porous structure. A further important property is their resistance against alkaline conditions, which is of great importance in large scale separation processes requiring frequent regeneration and sterilisation of the gel. Polysaccharide gels, unlike many other separation materials, allow in situ regeneration with alkaline agents, e.g. sodium hydroxide.
In many chromatography methods, such as gel filtration, ion exchange chromatography and affinity chromatography, polysaccharide gels are preferred because of their inertness and well established derivatisation chemistry. However, polysaccharide gels also exhibit certain drawbacks, like the limited mechanical stability. This is not a real problem when comparatively large gel beads. (around 100 xcexcm) are used in traditional chromatography. The situation is different when attempts are made to increase the separation efficacy by the use of smaller gel beads (5-20 xcexcm). At flow rates optimal for diffusion, the pressure drop in the gel bed is so high that the polysaccharide beads will collapse.
As a solution to the problem how to increase the separation efficacy, polysaccharide gels which contain two types of pores, small diameter diffusion pores (micropores), and large diameter flow-trough pores (macropores or superpores), have been disclosed (WO 93/19115). Such xe2x80x9csuperporousxe2x80x9d gels are manufactured by a method wherein a water-based solution of the polysaccharide is mixed, under controlled stirring, with an essentially water-immiscible organic phase to form an oil-in-water emulsion, which when allowed to solidify forms a network of two continuous phases; an aqueous polysaccharide phase and an organic phase. The aqueous phase forms a solid matrix, while the organic phase forms a network of flow-through pores within the matrix. Simultaneously, conventional small diameter diffusion pores are formed in the aqueous phase.
In order to obtain polysaccharide beads, the oil-in-water emulsion is emulsified in a second organic phase to give a three-phase system, comprising polysaccharide droplets in the organic phase. When the temperature is decreased below the gelling temperature of the polysaccharide, beads having the said two types of pores are obtained. In addition to smaller pores (diffusion pores normally around 20 to 500 xc3x85), which are typical for polysaccharides, the formed gel thus also contains larger pores (xe2x80x9csuperporesxe2x80x9d or xe2x80x9cflow-through pores, normally between 5 and 100 xcexcm).
When this superporous material is packed in a chromatographic column, some of the flow will pass via the superpores. The biomolecules to be separated are thus transported also to the inner parts of the beads by convective flow, which is a faster way of transportation than diffusion. Only short distances will have to be covered by diffusion in the superporous material and the particles will therefore, in spite of a much larger particle size, be as effective as conventional polysaccharide beads, and still give rise to a much lower pressure drop over the gel bed.
However, the presently known method for producing superporous gels has the drawback that this kind of three-phase system is unstable and has to be immediately stabilised by cooling in order to form superporous beads. There is thus a need for an improved method for manufacture of superporous gels or beads, which provides increased stability during the emulsifying process and which allows for large-scale production.
It has surprisingly been found that superporous polysaccharide beads as defined above can be conveniently produced by a method in which the above-mentioned drawbacks can be minimized.
Consequently, in a first aspect this invention provides a method for producing porous polysaccharide beads, comprising
(a) mixing a water-based solution (aqueous solution) of a polysaccharide under controlled stirring with an essentially water-immiscible first organic phase to form an emulsion (emulsion 1) which when allowed to solidify separates into a network of two continuous phases;
(b) adding a second organic phase comprising an emulsifier I and an organic solvent to form a three-phase system;
(c) allowing the said three-phase system to emulsify (formation of emulsion 2); and
(d) decreasing the temperature below the gelling point of the said polysaccharide, thereby obtaining beads having two sets of pores as defined above.
The method is characterised in that emulsifier I is a water-insoluble polymer capable of stabilising emulsion 2.
The two-phase system obtained in step (a) (Emulsion 1) can be produced by methods known from WO 93/19115. Briefly, a water-based polysaccharide phase is mixed, under vigorous stirring, with an essentially water-immiscible organic phase, consisting of one or more components, usually an organic solvent preferably together with an emulsifier of the type stabilising xe2x80x9coil-in-waterxe2x80x9d systems (emulsifier II).
The skilled person will be able to determine by known methods whether Emulsion 1 will be suitable for preparation of superporous beads. Rapid tests are available for verifying that a given combination of features will give a system suitable for the preparation of the desired material. One test involves study of Emulsion 1 under a microscope. The crucial factor is that the two phases must form a network of two continuous phases upon cooling. Another test involves a function test of pore flow. A small sample of Emulsion 1 is solidified and a thin (about 1 mm) gel slice is prepared. The gel slice is placed on a supporting net and a water jet is directed to the gel surface. If suitable super pores are present, the water jet will readily displace the organic phase in the gel slice. The appearance of the gel slice will change from white to semi-transparent in this process.
Emulsifier I stabilises emulsion 2 by being capable of adsorbing to the droplets of emulsion 1 in the second organic phase, thereby preventing destabilisation of the droplets by diffusion of the inner oil phase to the outer oil phase.
There is a large number of water-insoluble polymers that can be used as emulsifier I. Each candidate to be used can be prechecked, for instance by running the experimental protocol outlined in the experimental part. The proper functioning of a candidate will give rise to beads with flow-through pores also after prolonged emulsification. Flow-through pores can be detected either by ESEM (Environmental Scanning Electron Microscopy) or simply by visual inspection of the beads. Visual inspection: Transparent beads means that flow-through pores are absent while xe2x80x9cmilkyxe2x80x9d beads means that flow-through pores are present.
Initially the following water-insoluble polymers were of potential interest: Polybutadiene, polyisoprene, polyisobutene, polyacrylates, polymetacrylates, poly(vinyl acetate), polystyrene, polypropene oxide, polytetrahydrofurane, polycarbonate, non-crystalline polyesters, polydimethylsiloxan, ethyl cellulose, cellulose ethers, PEG-polyhydroxystearic acid block copolymers (Hypermer), styrene-butadiene/isoprene block copolymers (Kraton), water-insoluble ethylene oxide-propylene oxide block copolymers (Pluronics), ethylene oxide-tetrahydrofuran block copolymers (Berol).
Preferably, emulsifier I is selected from ethyl celluloses or polyvinyl acetates or other polymers having similar balance between hydrophilicity and hydrophobicity.
Polymers used as emulsifier I, should preferably contain at least 20, such as at least 30, monomeric units. When the polymer is an ethyl cellulose or a polyvinylacetate, the molecular weight of the polymer should preferably be above 7000 Da.
The organic solvent used in the first and second organic phase (step a and b, respectively) can e.g. be cyclohexane, toluene or heptane, or a mixture. Preferably, the said organic solvent is toluene. The organic solvent used in step a and step b may be the same or different.
It is an important aspect of the invention that the said three-phase system can be allowed to emulsify during a time period of more than 40 min, preferably around 1 hour, before the temperature is decreased below the gelling point of the said polysaccharide.
Different polysaccharides can be used in the methods according to the invention for production of superporous beads. Examples of suitable polysaccharides are agar, agarose, alginate, dextran, carrageenan, chitosan, cellulose and starch, as well as mixtures of these. The actual choice of polysaccharide will depend on the desired properties of the final product, for instance with regard to pore size, charge, stability in various media, cost, etc. Agarose is preferred, since it is essentially non-ionic and inert towards proteins.
If so required the polysaccharide is derivatized to have the proper solidifying/gelling properties.
As mentioned above, the obtained superporous beads have two sets of pores. The term xe2x80x9ctwo sets of poresxe2x80x9d is intended to mean that the formed polysaccharide beads comprise
(1) a first set of pores (superpores) essentially being above the size of diffusion pores, for instance between 5 and 100 xcexcm, and
(2) a second set of pores essentially being diffusion pores, for instance with a size between 20 and 521 xc3x85.
Typically the ratio between the pore diameters of the first set of pores and the particle diameter is in the interval 0.01-0.3, with preference for 0.05-0.2. The ratio between the pore diameters of the second set and the particle diameter may in the preferred variants extend up to 0.05 but is otherwise below 0.01.
The polysaccharide beads manufactured according to the invention have the same utility as the known gels disclosed in WO 93/19115. They can be produced in various shapes, e.g. more or less regular beads, and can be used in the manufacture of chromatographic media and as a carrier matrix for various biomolecules, like cells, enzymes, antibodies, etc. Because of its improved properties, superporous beads are particularly useful for high-performance liquid chromatography (HPLC).
After cooling the formed gel may be cross-linked and/or further derivatized in the same manner as described in WO 93/19115. Further derivatization encompasses among others that affinity groups are attached to the beads. An affinity group is a member of an affinity pair. Well-known affinity pairs are
(a) positively and negatively entities (ion exchange; the immobilised entity being selected among primary, secondary, tertiary and quaternary ammonium, sulphonate, sulphate, phosphonate, phosphate, carboxy etc groups),
(b) antibodies and antigens/haptens,
(c) lectins and carbohydrate structures,
(d) IgG binding proteins and IgG,
(e) pair of hydrophobic groups,
(f) polymeric chelators and chelates,
(g) complementary nucleic acids, etc.
Affinity members also include entities participating in catalytic reactions, for instance enzymes, enzyme substrates, cofactors, cosubstrates etc: Members of affinity pairs include chemically produced mimetics of biomolecules.
Potentially affinity groups may also be attached to the polysaccharide before it is used for preparing beads according to the invention.